Pre-prac Day 2 - SDS-PAGE gel interactive

Question 1

What does SDS-PAGE stand for?

Answer 1

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis

Question 2

What does the inner chamber in an SDS-PAGE set up contain?

Answer 2

2 gels or one gel and a “buffer dam” (the polyacrylamide gel is clamped to the inner chamber
2 electrodes (pos and neg)

Question 3

What doe the outer tank in an SDS-PAGE set up contain?

Answer 3

the inner chamber

Question 4

Where is the polyacrylamide gel in an SDS-PAGE setup located?

Answer 4

between glass/plastic panes
larger pane = spacer
smaller pane = short plate

Question 5

Where is the buffer located in an SDS-PAGE set up? hint - where in relation to the gel?

Answer 5

On top of the gel between the two glass/plastic panes (spacer and short plate)

Question 6

How should you pipette your sample for an SDS-PAGE?

Answer 6

Onto i.e. against the spacer (to help stabilise)
Tip above the next available well and just beneath the surface of the buffer
Gentle pipetting so the sample sinks into the well

Question 7

The sample in an SDS-PAGE gel is mixed with a sample loading buffer. What makes up this buffer (4 things)?

Answer 7

SDS - sodium dodecyl-sulphate
beta-mercaptoethanol
glycerol
bromophenol blue dye

Question 8

What is the role of SDS in the smaple loading buffer for SDS-PAGE?

Answer 8

sodium dodecyl-sulphate - coats the protein in a neg charge (so can move when a charge is applied across the gel)
binds to hydrophobic regions of the protien providing a neg charge (because neg charged head sulphate group) and unfolds the protein

Question 9

What is the role of beta-mercaptoethonol in the sample loading buffer for SDS-PAGE?

Answer 9

Acts as a reducing agent to get rid of disulfide bonds. This further denatures the protein (important to denature the proteins because if not denatured => slower migration time and we want the only factor to affect migration time to be the size of the protein?)

Question 10

What is the role of glycerol in the sample loading buffer for SDS-PAGE?

Answer 10

Glycerol adds density to the sample which helps it to drop to the bottom of the loading well and helps prevent the sample from diffusing out the well whilst the rest of the wells are being filled

Question 11

What is the role of bromophenol blue dye in the sample loading buffer for SDS-PAGE?

Answer 11

Allows a means of tracking progress
i.e acts as a tracking dye

Question 12

What would be the outcome if the voltage was too slow in an SDS-PAGE run?

Answer 12

Leads to a slow run
=> Proteins diffuse into the gel causing less well-defined bands

Question 13

What would be the outcome if the voltage was too high in an SDS-PAGE run?

Answer 13

There is a risk of the polyacrylamide gel melting

Question 14

What is the usual range of voltages used for an SDS-PAGE gel run?

Answer 14

~40-180 V

Question 15

What are three indicators of a correct SDS-PAGE run?

Answer 15

• bubbles showing there is current running
• upper (stacking) layer has proteins bunched closely
• lower (separating) layer has proteins resolved according to their size

Question 16

What are the key differences in properties in the stacking and resolving gel layer in the polyacrylamide ge in SDS-PAGE?

What implications do these differences in properties have?

Answer 16

stacking = upper layer
- lower percentage of acrylamide and a lower pH (6.8)
resolving = lower layer
- higher percentage of acrylamide and a higher pH (8.8)

Forms a discontinuous buffer system and in upper => proteins stack closely (hence name) and in lower=> resolve according to size

Question 17

Where should the gel from SDS-PAGE be cut after a run?

Answer 17

Removing the wells and cut in the stacking layer (avoiding resolving layer)

Question 18

What is SDS-PAGE trying to achieve?

Answer 18

Obtain high resolution separation of complex proteins (separation by weight/mass)

Question 19

DONE